Project description:In this work, we quantified mRNA flow rates between subcellular compartments in mouse embryonic stem cells. Combining metabolic RNA labeling, biochemical cell fractionation and RNA sequencing with mathematical modeling we were able to determine the kinetic rates of nuclear pre-, nuclear mature, cytosolic and membrane mRNA derived from more than 9000 protein-coding genes. For more than 5000 genes, we additionally estimated the transcript elongation rate. For most of the transcripts, mature nuclear half-lives are the longest, suggesting nuclear retention to be the rate-limiting step in the mRNA life cycle. Genes encoding transcription factors and immediate early genes possess fast kinetic rates, specifically a short nuclear half-life. Differentially localized mRNAs exhibit distinct combinations of rate constants, suggesting modular control within subcellular compartments. We show that membrane stability is high for membrane-localized mRNA and that cytosolic stability is high for cytosol-localized mRNA. Genes encoding target signals, such as signal peptides or transmembrane domains, have low cytosolic and high membrane half-lives with only slight differences between target signals. Nuclear-encoded mitochondrial proteins show long nuclear mature half-lives and otherwise similar features of cytoplasmic kinetics that do not resemble co-translational targeting to the mitochondria.
Project description:Tetrabromobisphenol-A (TBBPA) is a brominated flame retardant (BFR) commonly used in electronics to meet fire safety standards and has the largest worldwide production of any BFR. TBBPA has been detected in human breast milk and maternal/cord serum, indicating exposure to mothers, fetuses, and breastfeeding newborns although exposure to fetuses and newborns is poorly understood. Pregnant or nursing Wistar Han IGS rats were administered [14C]-TBBPA in a single dose (25 mg/kg, 2.5 μCi/kg) and euthanized between 0.5&24 h post dose to determine disposition in pregnant and nursing rats and their pups. Systemic exposure was largely unchanged between 1&8 h post dose in pregnant rats; [14C]-radioactivity in blood varied only slightly between 0.5&8 h (2.6 ± 0.6 → 2.6 ± 0.8 nmol-eq/mL) but was below the limit of detection at 24 h with an absorption half-life of 16min and elimination half-life of 17 h. Cmax was observed at 30min in lactating rats and concentrations fell steadily through 8 h. Plasma from pregnant rats contained a mixture of TBBPA and TBBPA-conjugates at 30min but only metabolites in subsequent samples. TBBPA was not detected in lactating dam plasma in this study. Placental concentrations increased through 8 h while whole-fetus Cmax occurred at 2 h post dose. In lactating animals, liver, uterus, and mammary time-concentration curves lagged slightly behind blood-concentration curves. It was clear from these studies that TBBPA is available to both the developing fetus and nursing pup following maternal exposure, and nursing pups are continuously exposed via contaminated milk produced by their mother. This research was supported in part by the Intramural Research Program of NIH/NCI.
Project description:Tetrabromobisphenol A (TBBPA) is the brominated flame retardant with the largest production volume worldwide. NTP 2-year bioassays found TBBPA dose-dependent increases in uterine tumors in female Wistar Han rats; evidence of reproductive tissues carcinogenicity was equivocal in male rats. To explain this apparent sex-dependence, the disposition and toxicokinetic profile of TBBPA were investigated using female Wistar Han rats, as no data were available for female rats. In these studies, the primary route of elimination following [14C]-TBBPA administration (25, 250 or 1,000 mg/kg) was in feces; recoveries in 72 h were 95.7±3.5%, 94.3±3.6% and 98.8±2.2%, respectively (urine: 0.2-2%; tissues: <0.1). TBBPA was conjugated to mono-glucuronide and -sulfate metabolites and eliminated in the bile. Plasma toxicokinetic parameters for a 250 mg/kg dose were estimated based on free TBBPA, as determined by UV/radiometric-HPLC analyses. Oral dosing by gavage (250 mg/kg) resulted in a rapid absorption of compound into the systemic circulation with an observed Cmax at 1.5 h post-dose followed by a prolonged terminal phase. TBBPA concentrations in plasma decreased rapidly after an IV dose (25 mg/kg) followed by a long elimination phase. These results indicate low systemic bioavailability (F<0.05), similar to previous reports using male rats. Elimination pathways appeared to become saturated leading to delayed excretion after a single oral administration of the highest dose (1,000 mg/kg); no such saturation or delay was detected at lower doses. Chronic high exposures to TBBPA may result in competition for metabolism with endogenous substrates in extrahepatic tissues (e.g., SULT1E1 estrogen sulfation) resulting in endocrine disruption.
Project description:The precise wiring of the nervous system relies on neurons extending their processes at the right time and place to find their appropriate synaptic partner. The mechanisms that determine when and where neurons extend their neurites during synaptogenesis remains a central question in the field. In the present study, we developed a cell culture system coupled with live imaging to investigate the wiring mechanisms in the developing nervous system. We focused on horizontal cells which are interneurons in the mammalian outer retina known to synapse selectively to distinct photoreceptors. Our data shows cultured horizontal cells extend neurites in a similar manner as in vivo with horizontal cells isolated from young mice extending more complex processes compared to those from adult retinas. In addition, horizontal cells cultured alone do not extend neurites and require other retinal cells for neurite extension suggesting that there must be extrinsic cues that promote neurite outgrowth. Moreover, these extrinsic cues do not appear to be solely secreted factors as supernatant from wild-type retinas is not sufficient to promote neurite outgrowth. In summary, we established a new system that can be used to decipher the mechanisms involved in neuronal wiring of the developing central nervous system.
Project description:In this work, we quantified mRNA flow rates between subcellular compartments in mouse embryonic stem cells. Combining metabolic RNA labeling, biochemical cell fractionation and RNA sequencing with mathematical modeling we were able to determine the kinetic rates of nuclear pre-, nuclear mature, cytosolic and membrane mRNA derived from more than 9000 protein-coding genes. For more than 5000 genes, we additionally estimated the transcript elongation rate. For most of the transcripts, mature nuclear half-lives are the longest, suggesting nuclear retention to be the rate-limiting step in the mRNA life cycle. Genes encoding transcription factors and immediate early genes possess fast kinetic rates, specifically a short nuclear half-life. Differentially localized mRNAs exhibit distinct combinations of rate constants, suggesting modular control within subcellular compartments. We show that membrane stability is high for membrane-localized mRNA and that cytosolic stability is high for cytosol-localized mRNA. Genes encoding target signals, such as signal peptides or transmembrane domains, have low cytosolic and high membrane half-lives with only slight differences between target signals. Nuclear-encoded mitochondrial proteins show long nuclear mature half-lives and otherwise similar features of cytoplasmic kinetics that do not resemble co-translational targeting to the mitochondria.
Project description:Current knowledge of the disposition kinetics of endogenous metabolites is founded almost entirely on poorly time-resolved experiments in which samples are removed from the body for later, benchtop analysis. Here, in contrast, we describe real-time, seconds-resolved measurements of plasma phenylalanine collected in situ in the body via electrochemical aptamer-based (EAB) sensors, a platform technology that is independent of the reactivity of its targets and thus is generalizable to many. Specifically, using indwelling EAB sensors, we have monitored plasma phenylalanine in live rats with a few micromolar precision and a 12 s temporal resolution, identifying a large-amplitude, few-seconds phase in the animals' metabolic response that had not previously been reported. Using the hundreds of individual measurements that the approach provides from each animal, we also identify inter-subject variability, including statistically significant differences associated with the feeding status. These results highlight the power of in vivo EAB measurements, an advancement that could dramatically impact our understanding of physiology and provide a valuable new tool for the monitoring and treatment of metabolic disorders.
Project description:Microbial metabolism drives biogeochemical fluxes in virtually every ecosystem. Modeling these fluxes is challenged by the incredible diversity of microorganisms, whose kinetic parameters are largely unknown. In poorly mixed systems, such as stagnant water columns or sediments, however, long-term bulk microbial metabolism may become limited by physical transport rates of substrates across space. Here we mathematically show that under these conditions, biogeochemical fluxes are largely predictable based on the system's transport properties, chemical boundary conditions, and the stoichiometry of metabolic pathways, regardless of the precise kinetics of the resident microorganisms. We formalize these considerations into a predictive modeling framework and demonstrate its use for the Cariaco Basin subeuphotic zone, one of the largest anoxic marine basins worldwide. Using chemical concentration data solely from the upper boundary (depth 180 m) and lower boundary (depth 900 m), but without a priori knowledge of metabolite fluxes, chemical depth profiles, kinetic parameters, or microbial species composition, we predict the concentrations and vertical fluxes of biologically important substances, including oxygen, nitrate, hydrogen sulfide, and ammonium, across the entire considered depth range (180-900 m). Our predictions largely agree with concentration measurements over a period of 14 years ([Formula: see text] = 0.78-0.92) and become particularly accurate during a period where the system was near biogeochemical steady state (years 2007-2009, [Formula: see text] = 0.86-0.95). Our work enables geobiological predictions for a large class of ecosystems without knowledge of kinetic parameters or geochemical depth profiles. Conceptually, our work provides a possible explanation for the decoupling between microbial species composition and bulk metabolic function, observed in various ecosystems.
Project description:In eukaryotic organisms, DNA replication is initiated at a series of chromosomal locations called origins, where replication forks are assembled proceeding bidirectionally to replicate the genome. The distribution and firing rate of these origins, in conjunction with the velocity at which forks progress, dictate the program of the replication process. Previous attempts at modeling DNA replication in eukaryotes have focused on cases where the firing rate and the velocity of replication forks are homogeneous, or uniform, across the genome. However, it is now known that there are large variations in origin activity along the genome and variations in fork velocities can also take place. Here, we generalize previous approaches to modeling replication, to allow for arbitrary spatial variation of initiation rates and fork velocities. We derive rate equations for left- and right-moving forks and for replication probability over time that can be solved numerically to obtain the mean-field replication program. This method accurately reproduces the results of DNA replication simulation. We also successfully adapted our approach to the inverse problem of fitting measurements of DNA replication performed on single DNA molecules. Since such measurements are performed on specified portion of the genome, the examined DNA molecules may be replicated by forks that originate either within the studied molecule or outside of it. This problem was solved by using an effective flux of incoming replication forks at the model boundaries to represent the origin activity outside the studied region. Using this approach, we show that reliable inferences can be made about the replication of specific portions of the genome even if the amount of data that can be obtained from single-molecule experiments is generally limited.
Project description:2,4,6-tribromophenol (TBP, CAS No. 118-79-6) is widely used as a brominated flame retardant and wood antifungal agent. TBP is frequently detected in environmental matrices, biota, and humans. In female SD rats, systemically available TBP (10 µmol/kg, IV) was rapidly excreted primarily via urine, with approximately 61% of the dose recovered after 4 h, and 89%-94% in 24 h; 5% was recovered in feces; and 1%-2% in blood/tissues. TBP administered to female SD rats (0.1-1000 µmol/kg) by gavage was well absorbed, with approximately 25% eliminated via urine after 4 h and approximately 88% after 24 h. Approximately 11% of a single oral dose was recovered in bile. Male SD rats and B6C3F1/J mice of both sexes had similar disposition profiles when administered a single oral dose of TBP (10 µmol/kg). Following administration, fecal recoveries varied only slightly by dose, sex, or species. TBP readily passed unchanged through both human (ex vivo only) and rat skin with between 55% and 85% of a 100 nmol/cm2 passing into or through skin. Concentrations of TBP in blood fit a two-compartment model after IV-dosing and a one-compartment model after oral dosing. Urine contained a mixture of TBP, TBP-glucuronide, and TBP-sulfate. Fecal extracts contained only parent TBP whereas bile contained only TBP-glucuronide. TBP did not appear to bioaccumulate or alter its own metabolism after repeated administration. TBP was readily absorbed at all doses and routes tested with an oral bioavailability of 23%-27%; 49% of TBP is expected to be dermally bioavailable in humans. From these data, we conclude that humans are likely to have significant systemic exposure when TBP is ingested or dermal exposure occurs.
Project description:Paclitaxel is a commonly used drug in the treatment of multiple solid tumors, including cancers of the breast, lung, and ovaries. Despite the established exposure-pharmacodynamic relationships for paclitaxel, treatment is associated with wide interindividual pharmacokinetic variability that leads to unpredictability of the agent's clinical activity and toxicity. We hypothesized that physiologically-based modeling approaches could be employed to predict the human pharmacokinetics of paclitaxel following administration of the approved Cremophor-based formulation (Taxol). The model was developed from tissue distribution studies performed in mice and applied to plasma concentration-time data obtained in adult cancer patients receiving Taxol at the approved dose and schedule (175 mg/m2 by a 3-hour intravenous infusion), taking into account interspecies differences in physiological parameters. The final model adequately captured the observed concentrations in patients and allowed prediction of paclitaxel distribution profiles in multiple target organs and can be applied to further refine the chemotherapeutic treatment with a clinically important agent.